Vasoactive intestinal peptide is known to play critical roles in modulating the intracellular and extracellular events involved in homeostasis, and are intimately involved in all major cognitive and non-cognitive homeostatic systems (Schofl et al., 1994). The multiple biological activities of peptides has led to extensive research focused on the exploitation of these peptide hormones as therapeutic drugs. Multiple replacements have been used to avoid the susceptibility of the amide bond to proteolytic cleavage. These include the use of nonstandard amino acids like D-amino acids, N-alkyl and N-hydroxy-amino acids, α-aza amino acids, thioamide linkage, design of peptide mimetics and prodrugs as well as amide bond modifications under the pseudopeptide linkage rubric (Dutta, 1993; Pasternak et al., 1999). Another approach has been the blockage of N-terminus or C-terminus of the peptide by acylation or amidation.
Vasoactive intestinal peptide is a 28-amino acid neuropeptide, which was first isolated from the porcine intestine (Said and Mutt, 1970). It bears extensive homology to secretin, peptide histidine isoleucine (PHI) and glucagon. The amino acid sequence for VIP is
(SEQ ID NO: 1)His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2.
VIP is known to exhibit a wide variety of biological activities in the autocrine, endocrine and paracrine functions in living organisms (Said, 1984). In the gastrointestinal tract, it has been known to stimulate pancreatic and biliary secretions, hepatic glycogenesis as well as the secretion of insulin and glucagon (Kerrins and Said, 1972; Domschke et al., 1977). In the nervous system it acts as a neurotransmitter and neuromodulator, regulating the release and secretion of several key hormones (Said, 1984). In recent years, attention has been focussed on the function of VIP in certain areas of the CNS as well its role in the progression and control of neoplastic disease (Reubi, 1995).
The importance of peptide growth factors and regulatory hormones in the etiology and pathogenesis in several carcinomas has long been recognized. Data from epidemiological and endocrinological studies suggest that neuropeptides like VIP which are responsible for the normal growth of tissues like the pancreas can also cause conditions for their neoplastic transformation (Sporn et al., 1980). Several lines of evidence indicate that VIP acts as a growth factor and plays a dominant autocrine and paracrine role in the sustained proliferation of cancer cells (Said, 1984). The stimulatory effect of VIP on tumor growth can be mediated directly by its receptors on cell membranes or indirectly by potentiation of the activities of other growth factors in tumor cells (Scholar et al., 1991). The synergistic effect of VIP and related pituitary adenylate cyclase activating polypeptide (PACAP) in glioblastomas is an illustration to the above fact (Moody et al., 1996).
The multiple physiological and pharmacological activities of VIP are mediated by high affinity G-protein coupled transmembrane receptors on target cells (Reubi et al., 1996). VIP receptors are coupled to cellular effector systems via adenyl cyclase activity (Xia et al., 1996). The VIP receptor, found to be highly over expressed in neoplastic cells, is thought to be one of the biomarkers in human cancers (Reubi et al., 1996). High affinity VIP receptors have been localized and characterized in neoplastic cells of most breast carcinomas, breast and prostate cancer metastases, ovarian, colonic and pancreatic adenocarcinomas, endometrial and squamous cell carcinomas, non small cell lung cancer, lymphomas, glioblastomas, astrocytomas, meningiomas and tumors of mesenchymal origin. Amongst, neuroendocrine tumors all differentiated and non-differentiated gastroenteropancreatic tumors, pheochromocytomas, small-cell lung cancers, neuroblastomas, pituitary adenomas as well tumors associated with hypersecretory states like Verner-Morrison syndrome were found to overexpress receptors for vasoactive intestinal peptide (Tang et al., 1997a & b; Moody et al., 1998a & b; Oka et al., 1998)). These findings suggest that new approaches for the diagnosis and treatment of these cancers may be based on functional manipulation of VIP activity, by designing suitable peptide derivatives of the same.
The present invention relates to the anti-neoplastic activity of novel VIP peptide analogs using selected constrained amino acids. These novel VIP analogs were found to bind to VIP receptor on cell membranes. The anti-neoplastic activity of the aforesaid peptides was also determined.
The design of conformationally constrained bioactive peptide derivatives has been one of the widely used approaches for the development of peptide-based therapeutic agents. Non-standard amino acids with strong conformational preferences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design. The conformational characteristics of α, α-dialkylated amino acids have been well studied. The incorporation of these amino acids restricts the rotation of φ, Ψ, angles, within the molecule, thereby stabilizing a desired peptide conformation. The prototypic member of α, α-dialkylated aminoacids, α-amino-isobutyric acid (Aib) or α, α-dimethylglycine has been shown to induce (β-turn or helical conformation when incorporated in a peptide sequence (Prasad and Balaram, 1984, Karle and Balaram, 1990). The conformational properties of the higher homologs of α, α-dialkylated amino acids such as di-ethylglycine (Deg), di-n-propylglycine (Dpg), di-n-butylglycine (Dbg) as well as the cyclic side chain analogs of α, α-dialkylated amino acids such as 1-aminocyclopentane carboxylic acid (Ac5c), 1-aminocyclohexane carboxylic acid (Ac6c), 1-aminocycloheptane carboxylic acid (Ac7c) and 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad et al., 1995; Karle et al., 1995). α, α-dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad et al., 1996). The applicants are not aware of any prior art for the synthesis of novel peptide analogs, encompassed in the present invention. The present invention exploits the conformational properties of α, α-dialkylated amino acids for the design of biologically active peptide derivatives, taking VIP as the model system under consideration.